Antibodies are drawing attention as pharmaceuticals as they are highly stable in plasma (blood) and have few side effects. Of these, a number of IgG-type antibody pharmaceuticals are available on the market and many antibody pharmaceuticals are currently under development (Non-patent Documents 1 and 2).
Almost all antibody pharmaceuticals currently available on the market are of the IgG1 subclass. IgG1 type antibodies are expected to be useful as anti-cancer antibody pharmaceuticals since they can bind to Fcγ receptor and exert ADCC activity. However, when it comes to antibody pharmaceuticals intended for neutralizing biological activity of an antigen, the binding of the Fc domain to Fcγ receptor, which is important for effector functions such as ADCC, can cause unnecessary side effects, and thus it is preferable to eliminate such binding activity (Non-patent Document 3). Furthermore, since Fcγ receptor is expressed in antigen-presenting cells, molecules that bind to Fcγ receptor tend to be presented as antigens. It has been reported that immunogenicity is and can be enhanced by linking a protein or peptide to the Fc domain of IgG1 (Non-patent Document 4 and Patent Document 1). Interaction between the antibody Fc domain and Fcγ receptor is thought to be a cause of the serious side effects encountered in phase-I clinical trials of TGN1412 (Non-patent Document 5). Thus, binding to Fcγ receptor is considered unfavorable in antibody pharmaceuticals intended for neutralizing the biological activity of an antigen from the perspective of side effects and immunogenicity.
A method for impairing the binding to Fcγ receptor is to alter the subtype of the IgG antibody from IgG1 to IgG2 or IgG4; however, this method cannot completely inhibit the binding (Non-patent Document 6). One of the methods reported for completely inhibiting the binding to Fcγ receptor is to artificially alter the Fc domain. For example, the effector functions of anti-CD3 antibodies and anti-CD4 antibodies cause side effects. Thus, amino acids that are not present in the wild type sequence were introduced into the Fcγ-receptor-binding domain of Fc (Non-patent Documents 3 and 7), and clinical trials are currently being conducted to assess anti-CD3 antibodies and anti-CD4 antibodies that have a mutated Fc domain and do not bind to Fcγ receptor (Non-patent Documents 5 and 8). Alternatively, Fcγ receptor-nonbinding antibodies can be prepared by altering the Fcγ R-binding domain of IgG1 (positions 233, 234, 235, 236, 327, 330, and 331 in the EU numbering; hereinafter abbreviated as position X (EU numbering)) to an IgG2 or IgG4 sequence (Non-patent Document 9 and Patent Document 2). However, these molecules contain new non-native peptide sequences of nine to twelve amino acids, which may constitute a T-cell epitope peptide and thus pose an immunogenicity risk. There is no previous report on Fcγ receptor-nonbinding antibodies that have overcome these problems.
Furthermore, for heterogeneity of the C-terminal sequence of an antibody, deletion of C-terminal amino acid lysine residue, and amidation of the C-terminal amino group due to deletion of both of the two C-terminal amino acids, glycine and lysine, have been reported (Non-patent Document 2). It is preferable to eliminate such heterogeneity when developing antibodies into pharmaceuticals.
Furthermore, in general, it is necessary that subcutaneous formulations are high-concentration formulations. From the perspective of stability and such, the concentration limit of IgG-type antibody formulations is generally thought to be about 100 mg/ml (Non-patent Document 13). Thus, it was a challenge to secure stability at high concentrations. However, there has been no report published on the improvement of the stability of IgG at high concentrations by introducing amino acid substitutions into its constant region. Meanwhile, instead of increasing the antibody concentration, methods that reduce the antibody dose by improving antibody kinetics in blood can be thought. A method for prolonging the antibody half-life in plasma has been reported and it substitutes amino acids in the constant region (Non-patent Documents 14 and 15); however, introduction of non-native sequences into the constant region is unpreferable from the perspective of immunogenicity risk.
Furthermore, physicochemical properties of antibody proteins, in particular, homogeneity, are very crucial in the development of antibody pharmaceuticals. For the IgG2 subtype, heterogeneity caused by disulfide bonds in the hinge region has been reported (Non-patent Documents 10, 16, 17, and 18 and Patent Document 3). It is not easy to manufacture them as a pharmaceutical in a large scale while maintaining differences of objective substance/related substance-related heterogeneity between productions. Thus, single substances are desirable as much as possible for antibody molecules developed as pharmaceuticals. In the present invention, differences in heterogeneity among productions can be understood, for example, as differences in heterogeneity among production lots. Heterogeneity in the production lots can be evaluated quantitatively by determining the diversity of molecular weight and structure of the produced antibody molecules.
As described above, it is desirable that the constant region sequences of antibody pharmaceuticals that are intended for neutralizing an antigen meet all the requirements in terms of the stability, C-terminal heterogeneity, immunogenicity (antigenicity), blood pharmacokinetics, and heterogeneity of hinge region. In particular, constant regions that do not have the heterogeneity of hinge region, which are more superior in blood pharmacokinetics than the natural constant regions such as of IgG1, are expected to be very useful as a constant region of antibody pharmaceuticals. However, altered constant regions that meet all of the above requirements have not yet been reported. Thus, there is a demand for antibody constant regions that have overcome the problems described above.
Documents of related prior arts for the present invention are described below.